Background: Anxiety disorder is one of the most common mental ailments exhibited by humans. It can cause considerable distress and debility. Anxiety is portrayed as a frame of mind concerned about future in association with preparation for possible, upcoming undesirable happenings. The present treatment for the disorder is having a lot of side-effects. An agent with good therapeutic effect and less side-effects is needed for the treatment of anxiety. Objectives: To investigate the anxiolytic activity of ethanolic extract of Moringa oleifera leaves in Swiss albino mice. Materials and Methods: The ethanolic extract of leaves of Moringa oleifera (200 mg/kg, i.p) was studied for its anxiolytic effect on Swiss albino mice by using Elevated Plus Maze (EPM) and Light Dark Arena (LDA) test. Results: The ethanolic extract of the leaves of Moringa oleifera (200 mg/kg, i.p) demonstrated significant (P < 0.001) anxiolytic activity in EPM and LDA models of anxiety. Conclusion: The data suggests that the ethanolic extract of Moringa oleifera leaves may have produced its anxiolytic effects via multiple mechanisms.

Anxiety is a feeling of nervousness, an unpleasant emotion due to the anticipation of nreal or threat imagined. The complexity of life in modern society frequently leads to varying degree of anxiety. One-eighth of the world population is suffering from anxiety. [1] Benzodiazepines (bzds), barbiturates, tricyclic antidepressants (TCAs) have been used for the treatment of anxiety disorders. [2] The side-effects of these drugs are sedation, rebound insomnia, withdrawal and tolerance, sexual dysfunction, muscle relaxation etc. [1] Therefore, an agent with good therapeutic effect and less side-effects is needed for the treatment of anxiety.

Moringa oleifera commonly known as drumstick tree in English; Subhanjana in Sanskrit; Saguna in Hindi; Sigru in Malayalam; [3] Nugga in Kannada; and Murungai in Tamil belongs to the family Moringaceae. [4] It's a soft wooded tree, and all the parts of the tree are edible. Traditionally, the leaves are used for the treatment of variety of disorder. Leaves are known to have anti-hyperglycemic, hepatoprotective, anti-hyperlipidemic, anti-microbial, anti-inflammatory, anti-convulsant, antioxidant properties. [5]

It is a natural anthelmintic, antibiotic, detoxifier, outstanding immune builder and is used in many countries to treat malnutrition and malaria. It is used in water purification and, therefore, helps in reducing the incidence of water-borne diseases. [6] Its a known fact that oxidative stress also plays an important role in the etiology of anxiety disorders. [7] This study is to investigate the effect of ethanolic extract of Moringa oleifera leaves in animal models of anxiety.

Materials and Methods

Animals

Young adult Swiss albino mice of either sex weighing 25-30 g were used in this study after obtaining Institutional Animal Ethical Committee Clearance. The mice were maintained under standard conditions in the animal house. The mice were kept in polypropylene cages and maintained on standard pellet diet and water ad libitum. Animals were acclimatized under standard laboratory condition and were kept in 12 hr day and night cycle for 7 days before conducting experiments.

Drugs / Dose / Route of administration

Diazepam (Cipla Ltd.) was obtained from Yenepoya Hospital Pharmacy in Mangalore. It was administered at a dose of (1 mg/ kg i.p).

Instruments

Soxhlet apparatus was used to prepare the plant extract. Elevated plus Maze apparatus and Light Dark Arena apparatus were used for screening the effect on anxiety.

Plant materials

Leaves of Moringa oleifera were used for the study. The fresh leaves were collected from Thiruvalla, Kerala. They were shade-dried. The dry leaves were grinded into coarse powder and extracted using Soxhlet apparatus. The leaves were authentified by Dr. Noeline. J. Pinto, Head of the Department, Botany, St. Agnes College, Mangalore, Karnataka, India.

Preparation of the extracts

Moringa oleifera ethanolic extract [MOEE]:

A weighed quantity (500 g) of the coarse powder was taken and extracted with ethanol (90%) in a Soxhlet apparatus. The extract was concentrated on a water bath at a temperature not exceeding 60°C. The ethanolic extract was suspended in distilled water. MOEE was administered at a dose of 200 mg/kg/bodyweight i.p.

Experimental design

Thirty-six animals were used in this study. The animals were divided into 3 groups. Each group consisting of 6 males and 6 females (n = 12).

Group-I: Normal Saline (0.1 ml i.p)

Group-II: MOEE (200 mg/kg i.p)

Group-III: Diazepam (1 mg/ kg i.p)

After an hour of the administration of test compounds, the animals were taken for the following tests for screening its anxiolytic activity.

To measure the level of anxiety in rodents, elevated plus maze was used. Three potential anxiogenic factors are open space, height, and novelty. The cross-shaped maze consists of 4 arms that are interconnected by a central platform. Two opposing arms are surrounded by side and end-walls (closed arms), whereas the remaining 2 arms are unprotected (open arms). The set-up consists of a maze of 2 open arms (16 cm × 5 cm), crossed with walls (12 cm high) and central platform (5 cm × 5 cm). The maze is suspended 25 cm above the room floor. The animal was placed on the central platform, facing one of the enclosed arms, and observed for 5 minutes (300 seconds). During the 5-min test period, the time spent in open and enclosed arms were recorded

The instrument consist of 2 parts, 1/3 with opaque walls and a cover (dark compartment), whereas the remaining 2/3 was open and illuminated (light compartment). The door between the two compartments permits mouse to move from one side to another. Each mouse was released in the light compartment and observed for 5 minutes. Time spent in light and dark compartment were recorded.

Statistical analysis

Results were expressed as mean ± SD. One-way analysis of variance (ANOVA) was carried out, and the statistical comparisons among the groups were performed with Tukey Krammer test using a statistical package program. P value < 0.001 was considered as significant.

Results

Elevated plus maze

MOEE-treated animals (Group II) and Diazepam-treated animals (Group III) showed a significant (P < 0.001) increase in the time spent in open arms [Table 1] by EPM test on comparing with the normal (Group I). There was significant difference in the time spent in the open arm between the MOEE-treated animals (Group II) and Diazepam-treated ones (Group III).

The above observations suggest that Moringa oleifera has anxiolytic activity.

Discussion

Anxiety is a frame of mind concerned about future in association with preparation for possible, upcoming undesirable happenings. [10] Neurotransmitters play an important role in the pathophysiology of anxiety disorders. [11]

It is a well-known fact that low level of Gamma Amino Butyric Acid (GABA) in CNS is most commonly linked with anxiety disorders. [12]

Serotonin (5-HT) has also an important role in the progress of anxiety disorders. Studies showed that patients with anxiety ailments have genetic polymorphisms in the 5-HT transporter. [13]

Apart from GABA and 5-HT, Dopamine and Norepinephrine (NE) also have an important role in the progress of anxiety disorders. Several animal studies including rodents and monkeys have suggested that reduced dopamine activity is associated with augmented anxiety. Several pre-clinical studies showed that there is an increased synthesis, release, and turnover of Norepinephrine in anxiety and stress, thus suggesting a role of NE in anxiety. This is further confirmed by the fact that agents which reduce the activity of NE in CNS have shown anti-anxiety effect. [14],[15]

Oxidative stress also plays an important role in the etiology of anxiety disorders. Anxiety disorders can also be due to free radical-induced damage to neurotransmitter system. [13],[16]

The results have shown that Moringa oleifera has anxiolytic property. Relatively very few information exist on the anxiolytic property of Moringa oleifera. The anxiolytic property can be due to its modulating role on the above neurotransmitters or due to its antioxidant property. Further studies are ongoing to find the active phytoconstituents present in the indigenous medicinal plant responsible for this anxiolytic property.